The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

HIV-1 accesses the nuclear DNA of interphase cells via a poorly defined process involving functional interactions between the capsid protein (CA) and nucleoporins (Nups). Here, we show that HIV-1 CA can bind multiple Nups, and that both natural and manipulated variation in Nup levels impacts HIV-1 infection in a manner that is strikingly dependent on cell-type, cell-cycle, and cyclophilin A (CypA). We also show that Nups mediate the function of the antiviral protein MX2, and that MX2 can variably inhibit non-viral NLS function. Remarkably, both enhancing and inhibiting effects of cyclophilin A and MX2 on various HIV-1 CA mutants could be induced or abolished by manipulating levels of the Nup93 subcomplex, the Nup62 subcomplex, NUP88, NUP214, RANBP2, or NUP153. Our findings suggest that several Nup-dependent ‘pathways’ are variably exploited by HIV-1 to target host DNA in a cell-type, cell-cycle, CypA and CA-sequence dependent manner, and are differentially inhibited by MX2.

Download Full-text

HIV-1 Cell-Free and Cell-to-Cell Infections Are Differentially Regulated by Distinct Determinants in the Env gp41 Cytoplasmic Tail

Journal of Virology ◽

10.1128/jvi.00655-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9324-9337 ◽

Cited By ~ 14

Author(s):

Natasha D. Durham ◽

Benjamin K. Chen

Keyword(s):

Amino Acids ◽

T Cells ◽

Point Mutations ◽

Cytoplasmic Tail ◽

Dependent Manner ◽

Cell Type ◽

Free Virus ◽

Cell Infection ◽

Hiv 1

ABSTRACTThe HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4+T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutations in the CT region that maintain or attenuate cell-free infection to investigate whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutations tested included truncations of structural motifs in the gp41 CT and two point mutations in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations, YW_SL and LL_RQ, severely impaired single-round cell-free infectivity 10-fold or more relative to wild-type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity 20-fold or more while resulting in a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the abilities of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell type dependent for large-CT-truncation mutants. Additionally, point mutants in LLP-3 can maintain multiround propagation from cell-to-cell in primary CD4+T cells.IMPORTANCEThe functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short-truncation mutants of ≤43 amino acids or mutants with point mutations in the LLP-3 helical domain of the CT, which are able to propagate via cell-to-cell infection in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cell-free and cell-to-cell infectionin vitroandin vivo.

Download Full-text

A Truncated Form of Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 Is Expressed in a Cell Type Dependent Manner

Virology ◽

10.1006/viro.1993.1651 ◽

1993 ◽

Vol 197 (2) ◽

pp. 751-756 ◽

Cited By ~ 69

Author(s):

Roger D. Everett ◽

Anne Cross ◽

Anne Orr

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Herpes Simplex Virus Type ◽

Dependent Manner ◽

Cell Type ◽

Truncated Form ◽

Early Protein ◽

A Cell ◽

Simplex Virus

Download Full-text

HIV envelope tail truncation confers resistance to SERINC5 restriction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101450118 ◽

2021 ◽

Vol 118 (21) ◽

pp. e2101450118

Author(s):

Tafhima Haider ◽

Xenia Snetkov ◽

Clare Jolly

Keyword(s):

T Cells ◽

Envelope Glycoprotein ◽

Cytoplasmic Tail ◽

Restriction Factor ◽

Viral Fusion ◽

Restriction Factors ◽

Complete Resistance ◽

Hiv Envelope ◽

Hiv 1

SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes.

Download Full-text

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics

10.1101/500975 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alba Torrents de la Peña ◽

Kimmo Rantalainen ◽

Christopher A. Cottrell ◽

Joel D. Allen ◽

Marit J. van Gils ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Transmembrane Domain ◽

Cytoplasmic Tail ◽

Full Length ◽

Broadly Neutralizing Antibodies ◽

Vaccine Research ◽

Similarities And Differences ◽

Membrane Tether ◽

Hiv 1

AbstractThe HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length native Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

Download Full-text

C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner

Cells ◽

10.3390/cells8101233 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1233 ◽

Cited By ~ 7

Author(s):

Leskelä ◽

Huber ◽

Rostalski ◽

Natunen ◽

Remes ◽

...

Keyword(s):

Neuroblastoma Cells ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Serum Starvation ◽

Dependent Manner ◽

Cell Type ◽

Proteasomal Activity ◽

N2a Cells ◽

Autophagy Induction ◽

A Cell

Dysfunctional autophagy or ubiquitin-proteasome system (UPS) are suggested to underlie abnormal protein aggregation in neurodegenerative diseases. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)-associated C9orf72 is implicated in autophagy, but whether it activates or inhibits autophagy is partially controversial. Here, we utilized knockdown or overexpression of C9orf72 in mouse N2a neuroblastoma cells or cultured neurons to elucidate the potential role of C9orf72 proteins in autophagy and UPS. Induction of autophagy in C9orf72 knockdown N2a cells led to decreased LC3BI to LC3BII conversion, p62 degradation, and formation of LC3-containing autophagosomes, suggesting compromised autophagy. Proteasomal activity was slightly decreased. No changes in autophagy nor proteasomal activity in C9orf72-overexpressing N2a cells were observed. However, in these cells, autophagy induction by serum starvation or rapamycin led to significantly decreased C9orf72 levels. The decreased levels of C9orf72 in serum-starved N2a cells were restored by the proteasomal inhibitor lactacystin, but not by the autophagy inhibitor bafilomycin A1 (BafA1) treatment. These data suggest that C9orf72 undergoes proteasomal degradation in N2a cells during autophagy. Lactacystin significantly elevated C9orf72 levels in N2a cells and neurons, further suggesting UPS-mediated regulation. In rapamycin and BafA1-treated neurons, C9orf72 levels were significantly increased. Altogether, these findings corroborate the previously suggested regulatory role for C9orf72 in autophagy and suggest cell type-dependent regulation of C9orf72 levels via UPS and/or autophagy.

Download Full-text

The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner

Viruses ◽

10.3390/v12060629 ◽

2020 ◽

Vol 12 (6) ◽

pp. 629 ◽

Cited By ~ 36

Author(s):

Mizuki Yamamoto ◽

Maki Kiso ◽

Yuko Sakai-Tagawa ◽

Kiyoko Iwatsuki-Horimoto ◽

Masaki Imai ◽

...

Keyword(s):

Cell Fusion ◽

Assay System ◽

Dependent Manner ◽

Cell Type ◽

S Protein ◽

Candidate Drug ◽

A Cell ◽

Transmembrane Serine Protease ◽

Nafamostat Mesylate

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 μM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat’s safety, make it a likely candidate drug to treat COVID-19.

Download Full-text